EP0018218A1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Interferon proteins and method of producing same
Assignee: SCHERING-PLOUGH Ltd
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Filing: 1980-04-18
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Granted: —
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Expires: 2000-04-18
Abstract
Abstract Purified human Le form interferon proteins are characterised and isolated by sodium dodecylsulphate polyacrylamide gradient electrophoresis as two major species of observed molecular weights 18 400 ± 200 daltons and 20 100 ± 200 daltons, and four minor species. Each individual pure interferon species has antiviral and anticellular activity, and each major species raises antibodies which neutralise the other. The pure interferon proteins are prepared from solutions thereof containing impurtities by antibody affinity chromatography optionally preceded by ligand affinity chromatography. Antibodies against the pure interferon proteins are useful in antibody affinity chromatography purification of crude interferon.
Inventors
- 👤 Kurt Frimann Berg
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 16:27
CPC Classifications
A; A61; A61K; A61K39/00; A61K39/395; A61K39/44; C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/555; C07K14/56; C07K16/00; C07K16/18; C07K16/24; C07K16/249; C12; C12P; C12P21/00; A61K38/00
Claims Analysis
Total Claims: 100
Claim 1
Independent Claim
95 words
Human Le form interferon proteins which under the SDS PAGE and staining conditions defined herein at a total interferon load of 0.9x10 6 IFU show two major sharp stained protein bands having antiviral interferon activity at 18,400 and 20,100 Daltons, respectively, and a minor stained protein band having 'antiviral interferon activity between 20,300 and 20,400 Daltons, together with smaller peaks of antiviral interferon activity at 19,500, 21,130, and 23,440 Daltons (said Dalton molecular weights being subject to an experimental accuracy of ±200 Daltons), said SDS PAGE acrylamide gradient showing essentially no other stained protein regions.
Claim 2
Dependent Claim
95 words
Human Le form interferon proteins which under the SDS PAGE and staining conditions defined herein at a total interferon load of 0.9x10 6 IFU show two major sharp stained protein bands having antiviral interferon activity at 18,400 and 20,100 Daltons, respectively, and a minor stained protein band having 'antiviral interferon activity between 20,300 and 20,400 Daltons, together with smaller peaks of antiviral interferon activity at 19,500, 21,130, and 23,440 Daltons (said Dalton molecular weights being subject to an experimental accuracy of ±200 Daltons), said SDS PAGE acrylamide gradient showing essentially no other stained protein regions.
Claim 3
Dependent Claim
108 words
Human Le form interferon proteins as claimed in claim 1 alternatively defined as proteins which under the SDS PAGE and staining conditions defined herein at a total interferon load of 3.8x10 IFU show six stained protein bands having antiviral interferon activity, viz. strong bands at 18,410 Daltons and 20,180 Daltons, respectively, a medium-strong band at 20,420 Daltons and just visible bands at 19,500 Daltons, 21,130 Daltons, and 23,440 Daltons, respectively (said Dalton molecular weights being subject to an experimental accuracy of ±200 Daltons), the peaks of antiviral interferon activity coinciding exactly with the stained protein bands, said SDS PAGE acrylamide gradient showing essentially no other stained protein regions.
Claim 4
Dependent Claim
25 words
Each individual protein having antiviral interferon activity which is a component of the human Le form interferon proteins claimed in claim 1 or claim 2.
Claim 5
Dependent Claim
16 words
A protein having antiviral interferon activity as claimed in claim 3 appearing at 18,400 ±200 Daltons.
Claim 6
Dependent Claim
16 words
A protein having antiviral interferon activity as claimed in claim 3 appearing at 20,100 ±200 Daltons.
Claim 7
Dependent Claim
37 words
A protein having antiviral interferon activty as claimed in claim 3 appearing between 20,300 and 20,400, at 19,500, at 21,130, or at 23,440 Daltons (said Dalton molecular weights being subject to an experimental accuracy of ±200 Daltons).
Claim 8
Dependent Claim
28 words
Proteins as claimed in claim 1 or 2 having a specific activity of at least 10 9 IFU per mg protein, as assessed by comparative SDS PAGE staining.
Claim 9
Dependent Claim
24 words
Proteins as claimed in claim 7 having a specific activity of 2x10 9 IFU per mg protein, as assessed by comparative SDS PAGE staining.
Claim 10
Dependent Claim
24 words
Protein(s) as claimed in any of the preceding claims, additionally characterized in that they exhibit anticellular activity and potentiate the Natural Killer cell system.
Claim 11
Dependent Claim
36 words
A protein as claimed in claim 4, additionally characterized in that it will neutralize antibodies raised against a protein as claimed in claim 5 or raised against proteins as claimed in claim 1 or claim 2.
Claim 12
Dependent Claim
36 words
A protein as claimed in claim 5, additionally characterized in that it will neutralize antibodies raised against a protein as claimed in claim 4 or raised against proteins as claimed in claim 1 or claim 2.
Claim 13
Dependent Claim
26 words
A protein as claimed in claim 3, additionally characterized in that it will neutralize antibodies raised against proteins as claimed in claim 1 or claim 2.
Claim 14
Dependent Claim
10 words
Human leukocyte interferon protein(s) as claimed in any preceding claim.
Claim 15
Dependent Claim
13 words
Human lymphoblastoid (Namalva) interferon Le form protein(s) as claimed in any preceding claim.
Claim 16
Dependent Claim
32 words
A formulation comprising a protein or proteins as claimed in any of the preceding claims, said formulation being adapted for administration to human beings or animals for prophylactic, therapeutic, or immunization effect.
Claim 17
Dependent Claim
14 words
A formulation as claimed in claim 15 adapted for parenteral, intranasal, or topical administration.
Claim 18
Dependent Claim
15 words
A formulation as claimed in claim 15 in the form of a stabilized aqueous solution.
Claim 19
Dependent Claim
27 words
A formulation as claimed in claim 17 in which the stabilizer is a protein or a combination of proteins which is non-toxic and non-immunogenic in human beings.
Claim 20
Dependent Claim
27 words
A formulation as claimed in claim 18 in which the stabilizer is selected from the group consisting of human serum proteins and fractions thereof, and human albumin.
Claim 21
Dependent Claim
33 words
20 A formulation as claimed in any of claims 15 - 19 in which the concentration of the pure interferon is in the range corresponding to 1 - 20 million IFU per ml.
Claim 22
Dependent Claim
16 words
A formulation as claimed in claim 17 for immunizing animals, in which the stabilizer is SDS.
Claim 23
Dependent Claim
18 words
A formulation as claimed in claim 21 which is buffered with PBS with a pH of about 7.2.
Claim 24
Dependent Claim
14 words
A formulation as claimed in claim 21 or 22 which additionally contains an adjuvant.
Claim 25
Dependent Claim
14 words
A formulation as claimed in claim 23 in which the adjuvant is Freund's adjuvant.
Claim 26
Dependent Claim
17 words
An SDS complex of a protein or proteins as claimed in any of claims 1 to 14.
Claim 27
Dependent Claim
12 words
An SDS complex as claimed in claim 25 isolated in solid form.
Claim 28
Dependent Claim
24 words
Antibodies raised against, or directed substantially only against, immunological determinants of human Le form interferon proteins claimed in any of claims 1 - 14.
Claim 29
Dependent Claim
47 words
Antibodies as claimed in claim 27 obtained by immunizing an immunizable animal with the 18,400 ±200, the 20,100 ±200, or a combination of the 18,400 ±200 and the 20,100 ±200 Daltons human Le form interferon protein component of the proteins claimed in claim 1 or claim 2.
Claim 30
Dependent Claim
44 words
Antibodies as claimed in claim 27 or 28 obtained by immunizing an immunizable animal with human Le form interferon protein or proteins as claimed in any of claims 1 - 14 obtained from the respective band or bands cut from the SDS PAGE gel.
Claim 31
Dependent Claim
28 words
Antibodies as claimed in claim 29 wherein the bands were cut from the SDS PAGE gel after staining of said gel and a short wash in distilled water.
Claim 32
Dependent Claim
37 words
A method for preparing antibodies as claimed in claim 27, comprising immunizing an immunizable animal against human Le form protein or proteins as claimed in any of claims 1 - 14 and obtaining antiserum from the animal.
Claim 33
Dependent Claim
27 words
A method as claimed in claim 31 in which the immunization is performed with a protein or proteins as claimed in any of claims 1 - 14.
Claim 34
Dependent Claim
48 words
A method as claimed in claim 32 in which the immunization is performed with the 18,400 ±200, the 20,100 ±200, or a combination of the 18,400 ±200 and the 20,100 ±200 Daltons human Le form interferon protein component of the proteins claimed in claim 1 or claim 2.
Claim 35
Independent Claim
31 words
A method as claimed in any of claims 31 - 33 in which the immunization is performed with a formulation as claimed in any of claims 17 and 21 - 24.
Claim 36
Dependent Claim
35 words
A method for producing antibodies as claimed in claim 27, comprising culturing a hybridoma cell clone producing antibodies directed against immunological determinants of human Le form interferon proteins and recovering antibodies from the culturing medium.
Claim 37
Dependent Claim
14 words
Antibodies when prepared by a method claimed in any of claims 31 - 35.
Claim 38
Dependent Claim
26 words
Antibodies as claimed in any of claims 27 - 30 or 36 (or fragments or derivatives thereof retaining the essential anti-interferon determinants) immobilized on a matrix.
Claim 39
Dependent Claim
12 words
3S. Antibodies as claimed in claim 37 covalently bound to the matrix.
Claim 40
Dependent Claim
18 words
Antibodies as claimed in claim 38 in which the matrix is a cross-linked agarose such as Sepharose 4B.
Claim 41
Dependent Claim
19 words
Matrix-immobilized antibodies as claimed in any of claims 37 - 39 which are substantially free from proteolytic enzymatic activity..
Claim 42
Dependent Claim
25 words
Matrix-immobilized antibodies as claimed in claim 40 which have been substantially freed from any proteolytic enzymatic activity by treatment with enzyme inhibitors or enzyme destructors.
Claim 43
Dependent Claim
28 words
Matrix-immobilized antibodies as claimed in claim 41 for which the treatment with enzyme inhibitors or enzyme destructors has been carried out with matrix-immobilized enzyme inhibitor or enzyme destructor.
Claim 44
Dependent Claim
34 words
Matrix-immobilized antibodies as claimed in claim 42 which, prior to their covalent binding to the matrix, have been passed through a column of matrix-immobilized poly-L-lysin and/or matrix-immobilized Soyabean Trypsin inhibitor, and/or matrix-immobilized kallikrein inactivator.
Claim 45
Dependent Claim
24 words
The use of antibodies as claimed in any of claims 27 - 30, and 36 - 43 for purifying human Le form interferon-containing solutions.
Claim 46
Dependent Claim
19 words
A protein having the significant interferon-characterizing determinants possessed by the proteins claimed in any of claims 1 - 14.
Claim 47
Dependent Claim
19 words
A protein having the significant immunological determinants possessed by the proteins claimed in any of claims 1 - 14.
Claim 48
Independent Claim
50 words
A method of producing human Le form interferon proteins as claimed in any of claims 1, 2, 7, and 8, comprising subjecting a solution containing human Le form interferon proteins to affinity chromatography such that said human Le form interferon proteins are selectively retained, and eluting said retained interferon proteins.
Claim 49
Dependent Claim
31 words
A method as claimed in claim 47, wherein the affinity chromatography comprises antibody affinity chromatography in which the antibodies are matrix-immobilized antibodies as claimed in any of claims 37 - 43.
Claim 50
Dependent Claim
28 words
A method as claimed in claim 48 in which at most 85% of the antibodies which were applied to the matrix at the immobilization stage became covalently bound.
Claim 51
Dependent Claim
37 words
A method as claimed in claim 48 or 49 in which the antibodies, prior to being bound to the matrix, had been substantially freed from any proteolytic enzymatic activity by treatment with enzyme inhibitors and/or enzyme destructors.
Claim 52
Dependent Claim
26 words
A method as claimed in claim 50 in which the removal of the proteolytic activity had been carried out with matrix-immobilized enzyme inhibitor and/or enzyme destructor.
Claim 53
Independent Claim
27 words
A method as claimed in any of claims 47 - 51 in which the solution applied to the antibody affinity matrix is a crude, unconcentrated interferon preparation.
Claim 54
Independent Claim
29 words
A method as claimed in any of claims 47 - 51 in which the solution applied to the antibody affinity matrix is a concentrated or partially purified interferon preparation.
Claim 55
Independent Claim
33 words
A method as claimed in any of claims 47 to 51 in which the interferon-containing solution comprises the interferon-containing protein fraction obtained by a protein precipitation treatment of a crude, unconcentrated interferon preparation.
Claim 56
Independent Claim
26 words
A method as claimed in 54 in which the protein precipitation treatment includes the addition of KSCN and the adjustment of the solution pH to 4.5.
Claim 57
Independent Claim
37 words
A method as claimed in any of claims 47 - 55 in which the solution applied to the antibody affinity matrix is an interferon solution which essentially only contains proteins in the 10,000 - 20,000 Daltons range.
Claim 58
Dependent Claim
53 words
A method as claimed in claim 56 in which the solution is the 10,000 - 20,000 Daltons eluate from a gel filtration performed with a buffer solution which contains about 25% by volume of a glycol such as ethylene glycol- and has an ionic strength corresponding to 1 M NaCl, pH about 7.2.
Claim 59
Dependent Claim
68 words
A method as claimed in any of claims 48 - 57 in which the human Le form interferon of the solution applied to the antibody affinity matrix is selected from the group consisting of human leukocyte interferons, human lymphoblastoid interferons, and proteins as claimed in claim 45 or 46, including such proteins when produced by cultivation of a microorganism carrying DNA coding for the production of such proteins.
Claim 60
Independent Claim
28 words
A method as claimed in any of claims 47 - 58 in which the affinity chromatography comprises a combination of ligand affinity chromatography and subsequent antibody affinity chromatography.
Claim 61
Dependent Claim
39 words
A method as claimed in claim 59 in which the ligand in the ligand chromatography is Cibacron F3GA or is another ligand capable of binding the human Le form interferon proteins according to the mechanism exerted by Cibacron F3GA.
Claim 62
Dependent Claim
33 words
A method as claimed in claim 60 in which the matrix-immobilized ligand is selected from the group consisting of Blue Dextran 2000, Blue Sepharosc Cl-6B , and Blue Dextran coupled to Sepharose 4B.
Claim 63
Dependent Claim
29 words
A method as claimed in claim 60 or 61 in which the ligand is immobilized on dextran of molecular weight of about 2 million Daltons, coupled to cross-linked agarose.
Claim 64
Dependent Claim
21 words
A method as claimed in claim 62 in which the matrix-immobilized ligand material is Blue Dextran coupled covalently to Sepharose 4B.
Claim 65
Independent Claim
96 words
A method as claimed in any of claims 60 - 63 in which the solution applied is an aqueous solution containing an Le form interferon in a form having a specific activity of at least 50,000 - 100,000 IFU per mg protein, the said solution being buffered to a pH of 6.5 - 8 and having an ionic strength substantially not exceeding the ionic strength of a 10 - 100 mM, in particular 20 mM, phosphate buffer, pH 7.2, solution, optionally together with water miscible organic solvent such as ethanol in amounts of 5 - 80%.
Claim 66
Dependent Claim
43 words
A method as claimed in claim 64 in which the elution is performed with an aqueous solution buffered to a pH of 6.5 - 8 and having an ionic strength corresponding to 0.5 - 0.7, in particular 0.5 - 0.65, molar NaCl solution.
Claim 67
Dependent Claim
26 words
A method as claimed in claim 65 in which the eluant has an ionic strength corresponding to 0.6 molar NaCl and is buffered to pH 7.2.
Claim 68
Independent Claim
26 words
A method as claimed in any of claims 64 - 66 in which the pH in the solution applied and in the eluate is about 7.2.
Claim 69
Dependent Claim
48 words
A method as claimed in claim 66 or 67 in which the interferon applied to the ligand affinity chromatography matrix has a specific activity of about 50,000 - 1,000,000, such as 100,000 - 1,000,000, e.g., 200,000 - 1,000,000, in particular about 500,000 - 1,000,000, IFU per mg protein.
Claim 70
Dependent Claim
68 words
A method as claimed in any of claims 60 - 68 in which the human Le form interferon of the solution applied to the antibody affinity matrix is selected from the group ccnsisting of human leukocyte interferons, human lymphoblastoid interferons, and proteins as claimed in claim 45 or 46, including such proteins when produced by cultivation of a microorganism carrying DNA coding for the production of such proteins.
Claim 71
Independent Claim
33 words
A method as claimed in any of claims 64 - 69 in which the interferon-containing solution comprises the interferon-containing protein fraction obtained by a protein precipitation treatment of a crude, unconcentrated interferon preparation.
Claim 72
Independent Claim
26 words
A method as claimed in 70 in which the protein precipitation treatment includes the addition of KSCN and the adjustment of the solution pH to 4.5.
Claim 73
Independent Claim
37 words
A method as claimed in any of claims 64 - 71 in which the solution applied to the antibody affinity matrix is an interferon solution which essentially only contains proteins in the 10,000 - 20,000 Daltons range.
Claim 74
Dependent Claim
53 words
A method as claimed in claim 72 in which the solution is the 10,000 - 20,000 Daltons eluate from a gel filtration performed with a buffer solution which contains about 25% by volume of a glycol such as ethylene glycol and has an ionic strength corresponding to 1 M NaCl, pH about 7.2.
Claim 75
Independent Claim
28 words
A method as claimed in any of claims 59 - 73 in which the eluate from the ligand affinity chromatography is loaded directly on the antibody affinity matrix.
Claim 76
Independent Claim
114 words
A method of purifying human interferon, comprising applying an aqueous solution containing human Le form interferon protein in a form having a specific activity of at least 50.000 - 100,000 IFU per mg of protein, the said solution being buffered to a pH of 6.5 - 8 and having an ionic strength substantially not exceeding the ionic strength of a 10 - 100 mM, in particular 20 mM phosphate buffer, pH 7.2 solution, optionally together with a water miscible organic solvent such as ethanol in amounts of 5 - 80%, on a matrix-immobilized ligand capable of binding the interferon according to the mechanism exerted by Cibacron Blue F3GA, and thereafter eluting interferon thus bound.
Claim 77
Independent Claim
114 words
A method of purifying human interferon, comprising applying an aqueous solution containing human Le form interferon protein in a form having a specific activity of at least 50.000 - 100,000 IFU per mg of protein, the said solution being buffered to a pH of 6.5 - 8 and having an ionic strength substantially not exceeding the ionic strength of a 10 - 100 mM, in particular 20 mM phosphate buffer, pH 7.2 solution, optionally together with a water miscible organic solvent such as ethanol in amounts of 5 - 80%, on a matrix-immobilized ligand capable of binding the interferon according to the mechanism exerted by Cibacron Blue F3GA, and thereafter eluting interferon thus bound.
Claim 78
Dependent Claim
26 words
A method as claimed in claim 75 when performed according to the conditions as defined in any of claims 61 - 63 and 65 - 74.
Claim 79
Dependent Claim
34 words
Human Le form interferon when prepared by the use of matrix-immobilized antibodies as claimed in any of claims 37 - 43 or by a method as claimed in any of claims 47 - 76.
Claim 80
Dependent Claim
40 words
Human Le form interferon as claimed in claim 77, having a specific activity of at least 30 1 10 6 IFU per mg protein, the protein determination being based on the Lowry procedure using pure human albumin serum as standard.
Claim 81
Dependent Claim
26 words
Human Le form interferon as claimed in claim 78 having a specific activity in the range from 30x10 6 to 10 8 IFU per mg protein
Claim 82
Dependent Claim
22 words
Human Le form interferon as claimed in claim 79 having a specific activity of 30x10 6 - 70x106 IFU per mg protein.
Claim 83
Independent Claim
49 words
A method of producing interferon proteins or proteins having the significant biological interferon-activity determinants thereof, comprising cultivating a microorganism carrying DNA coding for the production of Le form interferon proteins as claimed in any of claims 1 - 14 or 45, and recovering said proteins from the culture medium.
Claim 84
Dependent Claim
23 words
A method as claimed in claim 81 in which the DNA coding for the production of interferon proteins is present on a plasmid.
Claim 85
Dependent Claim
86 words
A method as claimed in claim 81 or 82 in which the DNA coding for the production of the interferon proteins has been prepared by treatment, with reverse transcriptase, of messenger RNA isolated from interferon-producing cells in a manner known per se and comprising the stage of antibody affinity chromatography and/or immunoprecipitation on a lysate of the cells, the antibody being used in the antibody affinity chromatography or in the immunc- precipitation being an antibody as claimed in any of claims 27 - 30 or 36.
Claim 86
Independent Claim
39 words
A method as claimed in any of claims 81 - 83 in which the interferon-producing clones of the microorganism have been selected by means of radio-labelled monospecific antibodies as claimed in any of claims 27 to 30 or 36.
Claim 87
Independent Claim
59 words
A method of removing proteins which are immunogenic in man from a protein solution which is to be administered to human beings, comprising subjecting the protein solution to an antibody affinity chromatography using antibodies against the immunogenic proteins, the antibodies used in the antibody affinity chromatography being of a kind which are non-immunogenic on parenteral administration to human beings.
Claim 88
Independent Claim
59 words
A method of removing proteins which are immunogenic in man from a protein solution which is to be administered to human beings, comprising subjecting the protein solution to an antibody affinity chromatography using antibodies against the immunogenic proteins, the antibodies used in the antibody affinity chromatography being of a kind which are non-immunogenic on parenteral administration to human beings.
Claim 89
Dependent Claim
24 words
A method as claimed in claim 85 in which the antibodies used in the antibody affinity chromatography are primate immunoglobulins or pig IgG immunoglobulins.
Claim 90
Dependent Claim
42 words
A method as claimed in claim 85 or 86 in which the protein solution subjected to the antibody affinity chromatography is an eluate from an antibody affinity chromatography using animal immunoglobulins which are immunogenic in man, such as sheep or rabbit immunoglobulins.
Claim 91
Dependent Claim
12 words
Matrix-immobilized antibodies for antibody affinity chromatography, the antibodies being pig IgG immunoglobulins.
Claim 92
Dependent Claim
12 words
Matrix-immobilized antibodies for antibody affinity chromatography, the antibodies being pig IgG immunoglobulins.
Claim 93
Dependent Claim
23 words
A process of purifying protein-containing solutions for administration to human beings by antibody affinity chromatography wherein the antibodies are matrix-immobilized pig IgG immunoglobulins.
Claim 94
Dependent Claim
23 words
A process of purifying protein-containing solutions for administration to human beings by antibody affinity chromatography wherein the antibodies are matrix-immobilized pig IgG immunoglobulins.
Claim 95
Independent Claim
62 words
A method of concentrating proteins from a protein-containing and SDS-containing aqueous solution, comprising precipitating SDS or a salt thereof from the solution to obtain a precipitate comprising a complex or complexes of SDS or a salt thereof with the protein, separating the precipitate from the solution, e.g. by centrifugation at 0 - 4°C, and redissolving the precipitate in a smaller liquid volume.
Claim 96
Independent Claim
62 words
A method of concentrating proteins from a protein-containing and SDS-containing aqueous solution, comprising precipitating SDS or a salt thereof from the solution to obtain a precipitate comprising a complex or complexes of SDS or a salt thereof with the protein, separating the precipitate from the solution, e.g. by centrifugation at 0 - 4°C, and redissolving the precipitate in a smaller liquid volume.
Claim 97
Dependent Claim
47 words
A method as claimed in claim 90 in which the precipitation of the SDS is obtained by a) lowering the temperature to 0°C for about 15 minutes, or b) adding a salt, e.g., a K + salt, which forms a precipitate with SDS or with SDS-protein complexes.
Claim 98
Dependent Claim
23 words
A method as claimed in claim 90 or 91 in which the protein-containing aqueous solution is a solution of pure or purified interferon.
Claim 99
Dependent Claim
21 words
A method as claimed in claim 92 in which the pure interferon is claimed in any of claims 1 - 14.
Claim 100
Independent Claim
37 words
A method as claimed in any of claims 90 - 93 in which SDS is present in the solution in a concentration of 0.1 - 4 per cent by weight, preferably about 0.1 per cent by weight.
References Cited (Backward Citations)
DE2722970A1
Cited
DE2812989A1
Cited
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